1. Field of the Invention
The present invention relates to the field of chemically modified forms of tumor necrosis factor (TNF) and uses thereof in the inhibition of tumor growth. In that the present invention presents novel forms of TNF, the invention also relates to methods of producing the modified TNF forms described. In that TNF is able to demonstrate anti-tumor cell activity, the present invention also provides methods of inhibiting tumor growth and treating TNF responsive cancers in humans.
2. Background of the Invention
Tumor necrosis factor (TNF) has become the recent focus of intense interest because of evidence for its role in a wide range of physiological mechanisms, immunopathologies, immunomodulation, and as a potential anti-neoplastic agent..sup.20 In the last few years following the cloning of its cDNA,.sup.21-24 many diverse studies have begun to elucidate features of its structure, including x-ray crystallography and development and characterization of variants with altered primary sequence..sup.26-31
Studies involving specific molecular modifications of the TNF molecule have proceeded despite scant prior evidence for the critical functional role of particular types of amino acid side-chains in TNF. Nevertheless, positively charged arginyl.sup.26 and lysyl.sup.25 residues have been shown or surmised to exert important effects on activity and/or three-dimensional structure. For example, introduction of arginyl residues as conservative or nonconservative substitutions in the N-terminal region of rHuTNF has been demonstrated to confer favorable effects on tumor cytotoxicity in vitro and to diminish toxicity in animal models..sup.26-28 Furthermore, lysyl residues have been proposed to participate in intra- and inter-molecular interactions with other amino acid side-chains in the rHuTNF trimer..sup.25
rHuTNF is a homotrimer of 17 kD subunits, each of which contains an N-terminal valine and six lysyl residues; two of these lysyl residues are known to be involved in intra- or intersubunit interactions..sup.25 TNF has been characterized as being cytotoxic for some tumor cell lines in vitro and as effective in causing necrosis of certain tumors in vivo..sup.12,32 This phenomenon was first described late in the last century when physicians noted rare spontaneous regressions of tumors in cancer patients.
However, TNF has also been shown to be a critical factor involved in the onset of septic shock..sup.12 In addition, TNF is identical to cachectin, a serum borne factor associated with cachexia, an emaciated condition of the body associated with chronic illness..sup.13,33 However, the tumor cytotoxic activity of TNF continues to prompt researchers to develop TNF preparations having reduced dose-limiting side effects with the greatest retention of tumor cytolytic activity.
For example, TNF has already become the subject of initial evaluation in Phase I/Phase II clinical trials at institutions worldwide..sup.34-37 However, the major impediment to further development remains in the described dose-limiting hypotension, perhaps-due to direct effects on vascular endothelium..sup.38-41 A strategy to better localize this cytokine in the tumor microenvironment and to diminish its systemic accessibility to normal tissue would thus provide a major advancement in the potential use of this valuable pharmaceutical agent in vivo.
Liposomes are emerging into early clinical evaluation as non-toxic drug carriers. They appear particularly well suited as carriers for hydrophobic drugs. At the same time, liposomes may target drugs directly to reticuloendothelial cell-rich organs, such as lung and liver, and indirectly to tumor beds via RES-mediated trafficking. The latter may be a particularly effective strategy in the use of TNF, as some reports indicate intratumoral administration is the most effective route.
However, native TNF demonstrates low encapsulation or association efficiencies for liposomes as reported in prior studies..sup.(5,6) For example, in the inventor's own laboratory, TNF encapsulation efficiencies were poor with native TNF and acylated TNF. In particular, native TNF bound liposomes with an efficiency of only 3.9% to preformed MLVs of PG/cholesterol, and from only 2.0-11.4% with ML's of mixtures of phosphatidylcholine (PC) phosphatidyl glycerol (PG), phosphatidyl serine (PS) and cholesterol (Chol). It is theorized that this poor affinity is due primarily to TNF's relatively low hydrophobicity.
More efficient methods of preparing liposome-associated TNF would be of significant medical value in the use of this agent as a therapeutic tool in the clinical management of cancer and other conditions, as well as greatly expand the scope of use to which TNF may be employed. Persons restricted from receiving TNF because of conditions potentially exacerbated by this agent may have TNF become available to them if the TNF hypotensive tendencies and other toxic side effects could be reduced and/or eliminated. More efficient methods for coupling TNF to liposomes pose a potential solution to reducing the toxic side effects of TNF.